Inhibition of metabotropic glutamate receptor 5 induces cellular stress through pertussis toxin-sensitive Gi-proteins in murine BV-2 microglia cells.

backgroundActivation of metabotropic glutamate receptor 5 (mGluR5) by (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) was shown to suppress microglia activation and decrease the release of associated pro-inflammatory mediators. In contrast, the consequences of mGluR5 inhibition are less well understood. Here, we used BV-2 cells, retaining key characteristics of primary mouse microglia, to examine whether mGluR5 inhibition by 2-methyl-6-(phenylethynyl)-pyridine (MPEP) enhances cellular stress and production of inflammatory mediators.

methodsBV-2 cells were treated with MPEP, followed by determination of cellular stress using fluorescent dyes and high-content imaging. The expression of inflammatory mediators, endoplasmic reticulum (ER)-stress markers and phosphorylated AMPKα was analyzed by quantitative PCR, ELISA and Western blotting. Additionally, phospholipase C (PLC) activity, cellular ATP content and changes in intracellular free Ca(2+) ([Ca(2+)]i) were measured using luminescence and fluorescence assays.

resultsTreatment of BV-2 microglia with 100 μM MPEP increased intracellular reactive oxygen species (ROS), mitochondrial superoxide, mitochondrial mass as well as inducible nitric oxide synthase (iNOS) and IL-6 expression. Furthermore, MPEP reduced cellular ATP and induced AMPKα phosphorylation and the expression of the ER-stress markers CHOP, GRP78 and GRP96. The MPEP-dependent effects were preceded by a rapid concentration-dependent elevation of [Ca(2+)]i, following Ca(2+) release from the ER, mainly via inositol triphosphate-induced receptors (IP3R). The MPEP-induced ER-stress could be blocked by pretreatment with the chemical chaperone 4-phenylbutyrate and the Ca(2+) chelator BAPTA-AM. Pretreatment with the AMPK agonist AICAR partially abolished, whilst the inhibitor compound C potentiated, the MPEP-dependent ER-stress. Importantly, the PLC inhibitor U-73122 and the Gi-protein inhibitor pertussis toxin (PTX) blocked the MPEP-induced increase in [Ca(2+)]i. Moreover, pretreatment of microglia with AICAR, BAPTA-AM, U-73122 and PTX prevented the MPEP-induced generation of oxidative stress and inflammatory mediators, further supporting a role for Gi-protein-mediated activation of PLC.

conclusionsThe results emphasize the potential pathophysiological role of mGluR5 antagonism in mediating oxidative stress, ER-stress and inflammation through a Ca(2+)-dependent pathway in microglia. The induction of cellular stress and inflammatory mediators involves PTX-sensitive Gi-proteins and subsequent activation of PLC, IP3R and Ca(2+) release from the ER.